The Rho GTPases are a structurally and functionally distinct GTPase of the Ras superfamily (Wennerberg et al., 2005, J. Cell Sci., 118:843-846; Takai et al., 2001, Physiol. Rev., 81:153-208). They are involved in a diverse array of cellular functions, including the regulation of actin and tubulin dynamics, cell polarity, membrane transport pathways and transcription factor activities (Ridley et al., 1992, Cell, 70:389-399; Braga et al., 1997, J. Cell Biol., 137:1421-1431; and Coso et al., 1995, Cell, 81:1137-1146). However, some members of the family (e.g., RhoBTB1, RhoBTB2 and RhoH) do not appear to be functionally closely related to the classical members such as RhoA, Rac1 and Cdc42 (Aspenstrom et al., 2004, Biochem J., 377:327-337). It is therefore accepted by those of skill in this area that the Rho GTPase of the Ras superfamily GTPases are most clearly defined by sequence homology.
When all the GTPase domains of the 150+ mammalian Ras superfamily members are aligned and are sorted into a dendrogram based on the alignment, the 20 Rho proteins form one separate branch of the Ras superfamily tree (Wennerberg et al., 2005, J. Cell Sci., 118:843-846). The overall homology within the GTPase domain of the Rho proteins separate them from the other Ras superfamily members. The Rho proteins share between 40-95% homology within the GTPase and they are 30% or less homologous to other Ras superfamily GTPases. In addition, the Rho proteins have one motif that is unique to this GTPase of small GTPases. This motif is called the Rho insert domain and is located between the fifth β strand and the fourth α helix in the small GTPase domain (Zong et al., 2001, Mol. Cell Biol., 21:5287-5298).
Accordingly, a small G-protein is considered a Rho family protein if, when aligned with other Ras superfamily proteins, it falls into the Rho branch (e.g., the protein has at least 40% homology to the other Rho proteins) and contains a Rho insert domain.
The Rho GTPases are known to be involved in the progress of many pathogenic processes such as metastatic invasion, bacterial and viral infections and hypertension (Symons, 1995, Curr. Op. Biotech., 6:668-674; Chen et al., 1996, Science, 274:2115-2118; and Uehata et al., 1997, Nature, 389:990-994). Because of their multiple roles in both basic cellular functions and in pathogenic processes there is much interest in developing assays that allow researchers to analyse the activity of Rho GTPases in cells. There is furthermore, great interest in developing assays that are compatible with high throughput screening applications for drug discovery targeting Rho GTPases or the Rho GTPase transduction pathways and for assays compatible with diagnostic applications.
The cellular activities mediated by Rho GTPases are dependent upon the activation state of the GTPase. When GTP is bound to Rho GTPases they are in an active state and are able to bind to effectors and propagate the signal cascade leading to a particular cellular response. When GDP is bound to Rho GTPases the Rho protein is inactive (Takai et al., 2001, Physiol. Rev., 81:153-208). Several assays have been developed that monitor the activation state of Rho GTPases.
One assay, the Rho effector pull-down assay, was originally developed for RhoA GTPases by Ren et al. (1999, EMBO J., 18:578-585) and for Rac1/Cdc42 GTPases by Benard et al. (1999, J. Biol. Chem., 274:13198-13204) and is the classical and most widely used assay. The method involves capture of activated Rho GTPase proteins by effectors bound to beads, release of the GTPase protein from the beads, separation of the beads from the released GTPase protein, followed by SDS-PAGE and analysis of the GTPase protein by western blotting. The assay suffers from poor reproducibility due to the multiple manipulations required in the performance of the assay, and low sensitivity. It is also not suitable for high throughput applications (Teusch et al., 2006, Assay and Drug Devel., 4:133).
There are several cell-based assays that use fluorescent bio-probes to detect activated Rho GTPases (Pertz et al., 2004, J. Cell Sci., 117:1313-1318). Several versions of this type of assay rely on a reporter system to monitor in vivo Rho GTPase activation. These cell-based assays, therefore, do not monitor the actual endogenous levels of the GTPase (Itoh et al., 2002, Mol. Cell Biol., 22:6582-6591; Pertz et al., 2006, Nature, 440:1069-1072; and Vadim et al., 2000, Science, 290:333-337). Other versions of cell-based assays use effector domains linked directly to an environmental dye to monitor endogenous in vivo GTPase activation. Because the placing of the environmental dye on any particular probe requires extensive analysis and a particular effector may not be compatible with dye linkage, the usefulness of any particular effector cannot be predicted (Nalbant et al., 2004, Science, 305:1615-1619). Furthermore, because the use of direct effector detection in vivo results in a probe that most often recognizes more than one GTPase, specificity is an issue in these assays. A further issue with this type of assay is that introduction of exogenous effector will actually alter the levels of Rho GTPase activation, which creates a technically challenging assay method (Pertz et al., 2004, J. Cell Sci., 117:1313-1318).
The fluorescent biosensor probes have also been applied to in vitro assays although their sensitivity is fairly low. Furthermore, that the dyes respond to environmental changes poses issues to drug screening applications (Hahn et al., U.S. Pat. No. 6,951,947 B2).
An enzymatic based method to detect Rho activation has been reported (Chen et al., 2003, J. Biol. Chem., 278:2807). The assay utilizes GST-effector-GBD to affinity precipitate active GTP-Rho. GTP is eluted and converted to ATP in a coupled enzymatic assay. ATP is then measured by the firefly luciferase method. This assay is highly dependent on the GST pull-down assay and, thus, has most of the drawbacks associated with this assay. Furthermore, since a Rho-specific antibody is not involved in this method, the specificity of the assay is limited.
An automated cell-based Rho activation assay has also been reported (Teusch et al., 2006, Assay and Drug Devel., 4:133). This assay was developed to replace the GST pull-down assay due to its incompatibility for high throughput screening and poor reproducibility. It is based on the ability of Rho to regulate the actin cytoskeleton. Since actin cytoskeleton is regulated by multiple signal pathways, the specificity for this assay is very limited.
Thus, there is a need for a Rho GTPase activation assay that is simple, specific for a particular GTPase protein, reproducible, sensitive and amenable to high throughput screening applications. The present invention provides for this need as well as others.